Urinary bladder cancer is the fifth most common cancer in the U.S. and the most expensive cancer to treat on a per patient basis because of its high incidence of recurrence and the lack of effective, targeted therapeutics. An expected 70,000 patients will be diagnosed with and 15,000 patients will die of urinary bladder cancer in the U.S. this year alone. Despite the high incidence and lack of therapy for bladder cancer, the mechanisms of tumor initiation and progression are largely unknown. The greatest risk factor for bladder cancer is smoking, which promotes malignant transformation of transitional epithelial cells of the bladder by mechanisms involving DNA damage and oncogene activation. Here we provide the first evidence for entirely novel signaling actions of Regulator of G protein Signaling 6 (RGS6) as an essential mediator of p53 induction during genotoxic stress and as a tumor suppressor in bladder. RGS6 is dramatically induced by tumorigenic stimuli including genotoxic stress and oncogene activation. RGS6 functions as an upstream activator of the ATM-p53- apoptosis pathway yet also induces apoptosis, arrests cell proliferation and blocks oncogenic transformation of cells by p53-independent mechanisms. These actions of RGS6 are independent of its canonical function as a heterotrimeric G protein in activator. We previously described a single nucleotide polymorphism in RGS6, which leads to increased RGS6 translation, which is associated with a significant reduction in the risk of bladder cancer in humans, particularly in smokers. Given these findings, it is especially significant that we observed a dramatically accelerated bladder carcinogenesis and loss of p53 induction in RGS6 null mice in response to their treatment with BBN, a DNA-damaging carcinogen derived from tobacco smoke that induces bladder carcinogenesis in mice closely mimicking that observed in the majority of human patients. The robust expression of RGS6 we found in bladder transitional epithelial cells was dramatically lost in patients with invasive bladder cancer. Our central hypothesis, formulated on the basis of compelling preliminary data, is that RGS6 is a novel master regulator of both DNA damage signaling and tumor suppression in bladder. Three specific aims are proposed: 1) Determine the importance of RGS6 as a tumor suppressor in a mouse model of bladder carcinogenesis and the role of p53 and ARF in its tumor suppressor function, 2) Determine the underlying molecular mechanisms by which RGS6 functions as an upstream modulator of DNA damage signaling and as a tumor suppressor in bladder transitional epithelial cells, and 3) Determine the molecular basis for RGS6 loss in human bladder cancer. These aims seek to elucidate the role of RGS6 in suppression of bladder tumorigenesis and the underlying mechanisms involved. Loss of RGS6, as observed in human bladder tumors, or its mutational inactivation would be expected to confer tumor cells with a predisposition to aberrant growth and enhanced survival, hallmarks of malignancy. The significance of these studies to human health is great. They will provide new understanding of the pathogenesis of urinary bladder cancer and potentially identify RGS6 as a biomarker for the prognosis or diagnosis of bladder cancer as well as a new therapeutic target for its treatment.